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Three series of analogs were regioselectively prepared from a protected forskolin precursor to afford 7-carbamoyl-7-desacetylforskolins (series 1),6-carbamoyl-7-desacetylforskolins (series 2),and 6-carbamoylforskolins (series 3). The analogs were pharmacologically evaluated for binding (IC50) to and activation (EC50) of type I adenylyl cyclase in membranes from stably transfected Sf9 cell lines expressing a single adenylate cyclase subtype. The following ranges were determined for the IC50's and EC50's of each individual series: series 1,IC50 = 43-1600 nM,EC50 = 0.5-9.6 microM; series 2,IC50 = 65-680 nM,EC50 = 0.63-6.5 microM; series 3,IC50 = 21-271 nM,EC50 = 0.5-8.1 microM (forskolin IC50 = 41 nM and EC50 = 0.5 microM). Activation paralleled binding; however,some analogs exhibited poor binding and good activation whereas others demonstrated good binding but poor activation. Steric bulk tended to diminish binding and activation when at the 6- or 7-position,although bulk was accommodated at the 6-position if the 7-site was reacetylated. Acylation of the 7-position by the carbamoyl linker or acetyl was important for obtaining good binding and activation; however,the effect was more pronounced with binding. For both binding and activation,small,linear,lipophilic substituents (propyl,allyl,isopropyl) are well tolerated at the 7-position but less so in the 6-position,even when the 7-site is reacetylated. Planar aromatic moieties (phenyl and 2-pyridinyl) demonstrated moderate to good potency for binding and activation when located at either the 6- or 7-positions. There is an overall trend toward increasing potency for both binding and activation with polar substituents. View Publication

The classical definition of lymphohematopoietic stem cells (LHSC),the most primitive progenitors of all blood cells,requires that they have the capacity for self-renewal and for the long-term production of all blood cell lineages. However,other characteristics of LHSC have been debated. Our previous data suggested that mouse LHSC are very slowly proliferating cells that generate delayed multilineage engraftment,while radioprotection" (rapid engraftment that will prevent early death from radiation-induced marrow aplasia) results from more committed progenitors. Alternatively� View Publication

A high proportion of the CD34+CD38- cells in normal human marrow are defined as long-term culture-initiating cells (LTC-IC) because they can proliferate and differentiate when co-cultured with cytokine-producing stromal feeder layers. In contrast,very few CD34+CD38- cells will divide in cytokine-containing methylcellulose and thus are not classifiable as direct colony-forming cells (CFC),although most can proliferate in serum-free liquid cultures containing certain soluble cytokines. Analysis of the effects of 16 cytokines on CD34+CD38- cells in the latter type of culture showed that Flt3-ligand (FL),Steel factor (SF),and interleukin (IL)-3 were both necessary and sufficient to obtain an approximately 30-fold amplification of the input LTC-IC population within 10 d. As single factors,only FL and thrombopoietin (TPO) stimulated a net increase in LTC-IC within 10 d. Interestingly,a significantly increased proportion of the CFC produced from the TPO-amplified LTC-IC were erythroid. Increases in the number of directly detectable CFC of textgreater 500-fold were also obtainable within 10 d in serum-free cultures of CD34+CD38- cells. However,this required the presence of IL-6 and/or granulocyte/colony-stimulating factor and/or nerve growth factor beta in addition to FL,SF,and IL-3. Also,for this response,the most potent single-acting factor tested was IL-3,not FL. Identification of cytokine combinations that differentially stimulate primitive human hematopoietic cell self-renewal and lineage determination should facilitate analysis of the intracellular pathways that regulate these decisions as well as the development of improved ex vivo expansion and gene transfer protocols. View Publication

Mitogen-activated protein (MAP) kinase cascades represent one of the major signal systems used by eukaryotic cells to transduce extracellular signals into cellular responses. Four MAP kinase subgroups have been identified in humans: ERK,JNK (SAPK),ERK5 (BMK),and p38. Here we characterize a new MAP kinase,p38beta. p38beta is a 372-amino acid protein most closely related to p38. It contains a TGY dual phosphorylation site,which is required for its kinase activity. Like p38,p38beta is activated by proinflammatory cytokines and environmental stress. A comparison of events associated with the activation of p38beta and p38 revealed differences,most notably in the preferred activation of p38beta by MAP kinase kinase 6 (MKK6),whereas p38 was activated nearly equally by MKK3,MKK4,and MKK6. Moreover,in vitro and in vivo experiments showed a strong substrate preference by p38beta for activating transcription factor 2 (ATF2). Enhancement of ATF2-dependent gene expression by p38beta was approximately20-fold greater than that of p38 and other MAP kinases tested. The data reported here suggest that while closely related,p38beta and p38 may be regulated by differing mechanisms and may exert their actions on separate downstream targets. View Publication

Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations,wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA-dependent protein kinase (DNA-PKcs). We report here the identification of the site of interaction of wortmannin on the catalytic subunit of PI 3-kinase,p110alpha. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110alpha. Phosphatidylinositol-4,5-diphosphate,ATP,and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin,while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated,recombinant p110alpha were mapped with anti-wortmannin and anti-p110alpha peptide antibodies,thus limiting the target site within a 10-kDa fragment,colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs,therefore,by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays,indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast,an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family,including PI kinases and ATM-related genes,that play a central role in many physiological processes. View Publication

Acute lymphoblastic leukaemia (ALL) is the most common cancer of childhood. Despite the progress achieved in its treatment,20% of cases relapse and no longer respond to chemotherapy. The most common phenotype of ALL cells share surface antigens with very early precursors of B cells and are therefore believed to originate from this lineage. Characterization of the growth requirement of ALL cells indicated that they were dependent on various cytokines,suggesting paracrine and/or autocrine growth regulation. Because many cytokines induce tyrosine phosphorylation in lymphoid progenitor cells,and constitutive tyrosine phosphorylation is commonly observed in B-lineage leukaemias,attempts have been made to develop protein tyrosine kinase (PTK) blockers of leukaemia cell growth. Here we show that leukaemic cells from patients in relapse have constitutively activated Jak-2 PTK. Inhibition of Jak-2 activity by a specific tyrosine kinase blocker,AG-490,selectively blocks leukaemic cell growth in vitro and in vivo by inducing programmed cell death,with no deleterious effect on normal haematopoiesis. View Publication

The bcr-abl oncogene,present in 95% of patients with chronic myelogenous leukemia (CML),has been implicated as the cause of this disease. A compound,designed to inhibit the Abl protein tyrosine kinase,was evaluated for its effects on cells containing the Bcr-Abl fusion protein. Cellular proliferation and tumor formation by Bcr-Abl-expressing cells were specifically inhibited by this compound. In colony-forming assays of peripheral blood or bone marrow from patients with CML,there was a 92-98% decrease in the number of bcr-abl colonies formed but no inhibition of normal colony formation. This compound may be useful in the treatment of bcr-abl-positive leukemias. View Publication

In cultures of embryonic striatum,we previously reported that EGF induces the proliferation of single precursor cells,which give rise to spheres of undifferentiated cells that can generate neurons and glia. We report here that,in vitro,these embryonic precursor cells exhibit properties and satisfy criteria representative of stem cells. The EGF-responsive cell was able to generate the three major phenotypes of the mammalian CNS--neurons,astrocytes,and oligodendrocytes. Approximately 90% of both primary spheres and secondary expanded clones,derived from the primary spheres,contained all three cell types. The increase in frequency of EGF-generated spheres,from 1% in primary culture to close to 20% in secondary culture,and the large number of clonally derived secondary spheres that could be generated from a single primary sphere indicate that EGF induces both renewal and expansion of the precursor cell itself. In population studies,the EGF-responsive cells were carried through 10 passages,resulting in a 10(7)-fold increase in cell number,without losing their proliferative and multilineage potential. Thus,this study describes the first demonstration,through clonal and population analyses in vitro,of a mammalian CNS stem cell that proliferates in response to an identified growth factor (EGF) and produces the three principal cell types of the CNS. View Publication

All-trans retinoic acid (ATRA) is successfully used in the cyto-differentiating treatment of acute promyelocytic leukemia (APL). Paradoxically,APL cells express PML-RAR,an aberrant form of the retinoic acid receptor type alpha (RAR alpha) derived from the leukemia-specific t(15;17) chromosomal translocation. We show here that AM580,a stable retinobenzoic derivative originally synthesized as a RAR alpha agonist,is a powerful inducer of granulocytic maturation in NB4,an APL-derived cell line,and in freshly isolated APL blasts. After treatment of APL cells with AM580 either alone or in combination with granulocyte colony-stimulating factor (G-CSF),the compound induces granulocytic maturation,as assessed by determination of the levels of leukocyte alkaline phosphatase,CD11b,CD33,and G-CSF receptor mRNA,at concentrations that are 10- to 100-fold lower than those of ATRA necessary to produce similar effects. By contrast,AM580 is not effective as ATRA in modulating the expression of these differentiation markers in the HL-60 cell line and in freshly isolated granulocytes obtained from the peripheral blood of chronic myelogenous leukemia patients during the stable phase of the disease. In NB4 cells,two other synthetic nonselective RAR ligands are capable of inducing LAP as much as AM580,whereas RAR beta- or RAR gamma-specific ligands are totally ineffective. These results show that AM580 is more powerful than ATRA in modulating the expression of differentiation antigens only in cells in which PML-RAR is present. Binding experiments,using COS-7 cells transiently transfected with PML-RAR and the normal RAR alpha,show that AM580 has a lower affinity than ATRA for both receptors. However,in the presence of PML-RAR,the synthetic retinoid is a much better transactivator of retinoic acid-responsive element-containing promoters than the natural retinoid,whereas,in the presence of RAR alpha,AM580 and ATRA have similar activity. This may explain the strong cyto-differentiating potential of AM580 in PML-RAR-containing leukemic cells. View Publication

The site-directed recombinase Cre can be employed to delete or express genes in cell lines or animals. Clearly,the ability to control remotely the activity of this enzyme would be highly desirable. To this end we have constructed expression vectors for fusion proteins consisting of the Cre recombinase and a mutated hormone-binding domain of the murine oestrogen receptor. The latter still binds the anti-oestrogen drug tamoxifen but no longer 17 beta-oestradiol. We show here that in embryonic stem cells expressing such fusion proteins,tamoxifen can efficiently induce Cre-mediated recombination,thereby activating a stably integrated LacZ reporter gene. In the presence of either 10 microM tamoxifen or 800 nM 4-hydroxy-tamoxifen,recombination of the LacZ gene is complete within 3-4 days. By placing a tamoxifen-binding domain on both ends of the Cre protein,the enzymatic activity of Cre can be even more tightly controlled. Transgenic mice expressing such an tamoxifen-inducible Cre enzyme may thus provide a new and useful genetic tool to mutate or delete genes at specific times during development or in adult animals. View Publication

Fumonisin B1 (FB1),a mycotoxin produced by the fungus Fusarium moniliforme,which is a common contaminant of corn,is suspected to be a cause of human esophageal cancer. FB1 is hepatotoxic and hepatocarcinogenic in rats,and although the mechanisms involved have not been clarified,the latter is associated with a weak initiating activity. The effects of FB1 on the activity of protein serine/threonine phosphatases (PPs) (PP1,PP2A,PP2B,PP2C and PP5/T/K/H) were investigated in the present study. Inhibition of dephosphorylation was noted for all five PPs with IC50 values of 80 microM-3000 microM. Among the five PPs examined,PP5 was most sensitive with an IC50 of 80 microM. This concentration is comparable to that estimated to be reached in the rat body by feeding FB1 to obtain hepatic tumors. Inhibition of PP5 could thus play important roles in the toxicity and carcinogenic action of FB1. View Publication

The synthetic cytokine (Synthokine) SC-55494 is a high-affinity interleukin-3 (IL-3) receptor ligand that stimulates greater in vitro multilineage hematopoietic activity than native IL-3,while inducing no significant increase in inflammatory activity relative to native IL-3. The aim of this study was to investigate the in vivo hematopoietic response of rhesus monkeys receiving Synthokine after radiation-induced marrow aplasia. Administration schedule and dose of Synthokine were evaluated. All animals were total-body irradiated (TBI) with 700 cGy 60Co gamma radiation on day 0. Beginning on day 1,cohorts of animals (n = 5) received Synthokine subcutaneously (SC) twice daily with 25 micrograms/kg/d or 100 micrograms/kg/d for 23 days or 100 micrograms/kg/d for 14 days. Control animals (n = 9) received human serum albumin SC once daily at 15 micrograms/kg/d for 23 days. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (NEUT; absolute neutrophil count [ANC] textless 500/microL) and thrombocytopenia (THROM; platelet count textless 20,000/microL) were assessed. Synthokine significantly (P textless .05) reduced the duration of THROM versus the HSA-treated animals regardless of dose or protocol length. The most striking reduction was obtained in the animals receiving 100 micrograms/kg/d for 23 days (THROM = 3.5 v 12.5 days in HSA control animals). Although the duration of NEUT was not significantly altered,the depth of the nadir was significantly lessened in all animal cohorts treated with Synthokine regardless of dose versus schedule length. Bone marrow progenitor cell cultures indicated a beneficial effect of Synthokine on the recovery of granulocyte-macrophage colony-forming units that was significantly higher at day 24 post-TBI in both cohorts treated at 25 and 100 micrograms/kg/d for 23 days relative to the control animals. Plasma pharmacokinetic parameters were evaluated in both normal and irradiated animals. Pharmacokinetic analysis performed in irradiated animals after 1 week of treatment suggests an effect of repetitive Synthokine schedule and/or TBI on distribution and/or elimination of Synthokine. These data show that the Synthokine,SC55 94,administered therapeutically post-TBI,significantly enhanced platelet recovery and modulated neutrophil nadir and may be clinically useful in the treatment of the myeloablated host. View Publication